The present invention relates to method and apparatus for counting particles by passing particles such as blood corpuscles through a minute aperture (minute orifice) and detecting and counting the particles.
An apparatus of this type for counting particles by passing particles such as blood corpuscles through a minute aperture, and detecting the electric charge (for example, the electric resistance) caused when the particles pass through the aperture is known. When measuring blood cells, the inside diameter of the aperture is about 50 to 100 .mu.m, and the length of the aperture is about 60 to 100 .mu.m. This aperture part is the sensitive zone in particle detection. The particle size is several microns.
In a particle counting apparatus of the electric resistance type, since the sensitive zone is broad, simultaneous passes may occur in the sample with many particles, and correction in the number counted is necessary. As the number of particles increases, simultaneous passes increase sharply. In this case, correction in the number counted is possible. But the magnitude of particle signals in simultaneous passes cannot be corrected, and as a result an error occurs in the distribution width of the particles in, and particle distribution or the like. In the electric resistance type, there is a favorable proportional relation between the size of the particle signal and the size of the actual particle. However, in case of a simultaneous pass, plural particle signals overlap to increase the signal size, and a particle signal proportional to the size of the particle is not obtained.
FIG. 3 is a particle size distribution diagram of erythrocytes (red blood corpuscles), in which the ordinate axis denotes the relative frequency. The solid line shows the distribution curve without a simultaneous pass, and the broken line represents the profile with a simultaneous pass. With a simultaneous pass, large particle signal components increase as mentioned above, and the obtained particle size distribution curve (broken line) is distorted.
Also known is an optical type particle counting apparatus for passing a liquid specimen through a minute aperture enclosed with sheath fluid (sheath flow), and detecting light signals from the particles. This is called a flow cytometer. In the optical type, as compared with the electric type, the sensitive zone is narrow, and simultaneous passes hardly occurs so that correction is not necessary. In this case, the sheath flow means a flow covered with a laminar flow liquid (sheath of liquid) around the particle suspension, the particles being arranging neatly in one row at high precision in the middle of the minute aperture to effect passing.
On the other hand, a combined apparatus of the electric type and the optical type is also known. By emitting light to the minute aperture in a direction orthogonal to the flow of particles, signals of scattered light and fluorescence can be detected simultaneously on individual particles. In this case, too, there are problems relating to the size of the particle signals due to simultaneous passes.
The following methods are known to solve the above problems.
(a) To preliminarily dilute the liquid specimen to such an extend that simultaneous passes cannot occur.
(b) To reduce the flow rate of liquid specimen in the sheath flow so as to pass a narrow flow of liquid specimen in the minute aperture.
The method of (a) takes time and labor in diluting the process. Besides, the number of particles is different in each specimen, and the dilution proportion is not known.
The method of (b) takes a long counting time.